AAV gene therapy to restore fertility in mammalian Sertoli cell dysfunction models

ABSTRACT:
Infertility impacts 48 million couples worldwide and 50% of cases are due to a male factor. Non-obstructive
azoospermia (NOA) is the absence of sperm due to spermatogenic failure. Some cases are due to known
conditions (Klinefelter’s Syndrome, Y chromosome microdeletions, and hypogonadism) but many cases are
idiopathic (unexplained). Recent identification of novel monogenic causes of NOA sparks the possibility of gene
therapy as a potential treatment option. There are ethical, legal and societal concerns about gene therapy
approaches that would modify the germline and potentially be passed to progeny. Therefore, development of
gene therapy approaches for male infertility should consider the risk of germline modification and transmission.
Sertoli cells are the only testicular somatic cells that are inside the seminiferous tubules and in direct contact
with the germ cells and Sertoli cell mutations can lead to NOA in both mice and humans. I will test the feasibility
and safety of in vivo Sertoli cell gene therapy in mouse models of human NOA. Adeno-associated virus (AAV)
is an optimal vector for Sertoli cell gene therapy because there are many AAV serotypes with cell type specificity
of infection, it can have long lasting expression, it has low toxicity in the testis, and does not typically integrate
into the genome. AAV has been used to introduce a corrective Kitl gene into Sertoli cells of Kitl-/- mice that have
an NOA phenotype, restoring spermatogenesis. In humans, mutations in the KITL gene do not cause infertility.
Under separate funding, our group has been performing whole genome or whole exome sequencing to identify
genetic variants in men with NOA. I have screened those data to identify NOA-associated variants in MAP7 and
CLCN2 that are expressed by Sertoli cells and cause NOA when knocked out in mice. I will use CRISPR/Cas9
gene editing to introduce the patient variants into the orthologous regions of the mouse genome to create new
mouse models of human NOA. I will use these models to test the hypothesis that, fertility can be safely restored
by AAV-mediated Sertoli cell gene therapy and that this can be accomplished without germline modification or
transmission to progeny. To facilitate the translation of this technology toward the human clinic, I will utilize a
novel human testis organ culture technique to identify AAV serotypes that exhibit Sertoli cell specific infection in
human testicular tissue. Completion of my project will demonstrate the safety and feasibility of Sertoli cell gene
therapy using AAV vectors. I will uncover new knowledge into genetic causes of Sertoli cell dysfunction and
propose a technique that can test other aspects of AAV use in human tissue. Overall, the completion of this grant
will greatly increase my technical research skills in gene therapy and male infertility in a bench to bedside
research pipeline. This will provide a strong foundation of knowledge and skills to prepare me for a career as a
faculty and independent researcher in academia.

Grant Number: 5F31HD114406-03
NIH Institute/Center: NIH
Principal Investigator: Georgia Rae Atkins